Effect of different methods of treating tissues on the yield of extracted RNA
Effect of different methods of treating tissues on the yield of extracted RNA
RNA Laboratory Techniques Manual (Molecular Cloning - Laboratory Guide Series)
Operation method
Effect of different methods of treating tissues on the yield of extracted RNA
Principle
One of the most important things to keep in mind during RNA extraction is how to suppress RNase-induced degradation of RNA while the tissue or cells are being completely lysed in the denaturing solution. When processing fresh tissue directly, it is important that all cells are exposed to the denaturant as quickly as possible to allow for complete lysis. Tissues that are difficult to process, such as hard tumors, bacterial cells, plant roots, etc., are generally not lysed very efficiently even with a homogenizer. In this case, the tissue can usually be processed by freezing it first to allow for adequate lysis. In order to freeze samples quickly enough to allow complete freezing of whole pieces of tissue, the tissue should therefore be cut into small pieces and then submerged in liquid nitrogen before freezing. This will freeze the tissue chunks in the fastest way possible. Alternatively, a metal disk can be placed on dry ice as a freezing surface to provide rapid freezing of pieces of tissue. Each of the following three force methods describes processing 0.lg frozen mouse liver tissue in three different ways to produce tissue/cell lysis products. The lysis products can be purified for RNA extraction or used to estimate the yield of pdy(A)+RNA. Although all three methods utilize the same guanidine buffer in order to thoroughly lyse the cells, they differ in the previous steps, i.e., how the tissue is processed or in how it is lysed. Move Method 1: Processing frozen tissue in a homogenizer For more product details, please visit Aladdin Scientific website.
Yield: 4.1ugpoly(A)ˉRNA
Frozen tissues were sliced into small pieces of approximately Cl5 cm2 on dry ice, transferred to a homogenizer, and continuously homogenized while slowly adding lysis buffer.
Method 2: Processing of frozen tissue by syringe.
Yield: 3.2ugpoly(A)-RNA.
Frozen tissue was cut into small pieces of approximately 0.5 cm2 on dry ice and repeatedly pumped ten times through an 18-gauge standard syringe needle while slowly adding lysis buffer.
Method 3: Tissue was ground into a powder in liquid nitrogen.
Yield: 7.1ugpoly(A)ˉ RNA.
The frozen sample was placed in a mortar and pestle for vigorous grinding to pulverize it. When the tissue has been ground into a powder, the Variable Pleasure Buffer is added to the mortar and stirred well. The mixture should be melted and transferred to a suitable container for further manipulation. When the tissue is ground to a powdered form in liquid nitrogen, cell lysis is more complete and therefore nearly double the yield of the previous two methods can be obtained.
It can be seen that even for N-types of tissues, the use of the same reagent for RNA extraction resulted in significant differences in the amount of RNA extracted simply due to differences in the method of processing the tissue samples. Therefore, when extracting RNA from different tissues, not only should the most appropriate extraction method be selected, but also the most appropriate tissue treatment method should be selected according to the treated tissues and the purpose of extraction, only in this way can the best results be obtained.